DESCRIPTION: This work involves general characterization of enzymes involved in respiration. The subject organism is the thermophilic bacterium Thermus thermophilus. With a working knowledge of its biology, biochemistry and molecular biology, it has been possible to carry out a wide range of biochemical, biophysical, and physical characterizations of its respiratory proteins. Because the respiratory proteins from this organism are functional and structural homologs of their eukaryotic counterparts, the results of our structure-function studies are directly applicable to all other such enzymes, notably those of the mammalian mitochondrion. During the past funding period we: (1) Determined the DNA sequence of structural genes encoding cytochromes caa3 and ba3, and by making detailed comparisons of their deduced amino acid sequences demonstrated that they are members of heme/Cu oxidase super-family. (2) We carried out a number of spectroscopic investigations with cytochromes caa3 and ba3 and of Rieske-type proteins, which led to detailed new information about the active site structures of these proteins. (3) Our most exciting discovery was that a small portion of the subunit II of cytochrome ba3 gene could be subcloned, expressed in E. coli, and was found to form a very stable CuA center. This discovery opens many new research opportunites for exploring the properties of this novel and somewhat unexpected arrangement of Cu atoms, and this is a very fast moving field of research. Accordingly, the current proposal is tightly focussed on just two scientific goals while the longer-term goals of the project are served under a much broader technical goal. The Specific Aims are: (1) Toward achieving a detailed understanding of CuA structure and function, we will assemble and interpret spectral, structural and functional information from a water-soluble, CuA containing protein. (2) As a first step toward understanding assembly of the CuA center, we will examine the properties of a yellow-colored intermediate that forms during CuA synthesis. (3) We will continue our efforts to develop a combined genetic, structural and kinetic 'system' that will permit detailed biophysical study of the cytochrome ba3. The significance of this work to human health lies in a deeper understanding of the enzymes that provide energy to all cells. That mutations in human respiratory proteins are being found at a prodigious rate, is evidenced by the appearance of a new web site, http://infinity.gen.emory.edu/mitomap.html.